Analysis of a zebra stripe double mutant
--Surdam, D; Bertrand-Garcia, R

Over the last several years we have been characterizing the zebra stripe mutants in maize. The recessive zebra stripe mutations, (zb1, zb2, zb3, zb4, and zb7), affect chloroplast development by reducing thylakoid membrane formation and the number of grana. Mutant plants typically express transverse yellow striping in the juvenile leaves of the plant and show wild type adult leaves.

Study of several zebra stripe double mutations in our lab has led to some very interesting results. We selfed heterozygous zebra stripe-3 (zb3)-zebra stripe-7 (zb7) mutant families, and recovered a double mutant phenotype.

Double mutants of zb7, zb3/zb7, zb3 segregated as 1/16 of the population; chi square analysis of 162 plants resulted in a x2=.107 giving us a p value =.975. The double mutants were phenotypically bleached-yellow, with little or no green pigment in the juvenile leaves (Fig. 1).

Adult double mutants were stunted compared to wild type and single mutant siblings (mutant adults averaging about 23.7 cm in height). Expressivity of the bleached yellow phenotype was most severe in the first five (juvenile) leaves. Adult leaves were successively greener.

Analysis of the double mutants by Western analysis revealed some fascinating results. Antibodies to four chloroplast photo-system complexes (Sub 4, psaD, psbA, and ATPase) were used to assess wild-type, zb3, zb7 and the zb3-zb7 double mutants. Wild type, zb7, zb3 and zb3-zb7 double mutants were collected, chloroplast membrane proteins isolated and protein concentration determined by spectrophotometry. The proteins were separated by SDS-PAGE electrophoresis using a 12% gel. The proteins were blotted to nitrocellulose, the membrane was probed with specific antibodies and binding detected using an Amersham ECL-Western blotting analysis kit.

The blot in Figure 2 shows the results for juvenile leaves collected from all four genotypes. The most striking result is the lack of psaD in the zb3-zb7 double mutant. psaD is a component of the photosystem I complex. All juvenile leaves of the double mutants revealed a lack of psaD. Overloading gels with 5X concentrations of chloroplast membrane proteins from the double mutant, and overexposure of films, still showed a lack of psaD. Analysis of adult zb3-zb7 double mutant leaves revealed a small amount of the psaD protein, suggesting that the juvenile leaves lack PSI completely but recover small amounts of PSI in the adult leaves.

 Figure 1. zb3-zb7 double mutant and wild type siblings.

 Figure 2. Lanes 1, 2, 3, and 4 represent the zb3-zb7 double mutation, zb7 mutation, zb3 mutation, and wild type chloroplast extracts, respectively. Rows A, B, C, and D represent the chloroplast photo-system complexes of Sub 4, psaD, psbA, and ATPase respectively. The arrow indicates the absence of psaD in the double mutant.

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